Production Of Pharmaceutical Proteins Research Articles

An ovalbumin fusion strategy to increase recombinant protein secretion in chicken eggs

Maternal secretion of recombinant proteins into chicken eggs may provide a viable approach for pharmaceutical production but remains limited by poor secretion efficiency through the membrane of oviduct cells, despite high expression levels. Here, we used site-specific integration of an EGFP fused to the OVAL gene by a rigid linker, (EAAAK)3, at the endogenous ovalbumin locus in chicken primordial germ cells to generate OVAL-E3-EGFP transgenic chickens, with transgenic chickens expressing CMV immediate enhancer/β-actin-driven EGFP (CAG-EGFP) as a non-secreted control. In OVAL-E3-EGFP chickens, EGFP protein produced in maternal oviducts accumulates to high levels in eggs, but not in eggs of CAG-EGFP chickens. These results indicated that the secretion of foreign proteins can be substantially increased through fusion to the highly secreted endogenous ovalbumin. This study describes a basis for high yield recombinant protein expression in chicken eggs, enabling rapid and scalable production of numerous pharmaceutical proteins or metabolites.

How genome editing changed the world of large animal research.

The first genetically modified large animals were developed in 1985 by microinjection to increase the growth of agricultural livestock such as pigs. Since then, it has been a difficult trail due to the lack of genetic tools. Although methods and technologies were developed quickly for the main experimental mammal, the mouse, e.g., efficient pronuclear microinjection, gene targeting in embryonic stem cells, and omics data, most of it was-and in part still is-lacking when it comes to livestock. Over the next few decades, progress in genetic engineering of large animals was driven less by research for agriculture but more for biomedical applications, such as the production of pharmaceutical proteins in the milk of sheep, goats, or cows, xeno-organ transplantation, and modeling human diseases. Available technologies determined if a desired animal model could be realized, and efficiencies were generally low. Presented here is a short review of how genome editing tools, specifically CRISPR/Cas, have impacted the large animal field in recent years. Although there will be a focus on genome engineering of pigs for biomedical applications, the general principles and experimental approaches also apply to other livestock species or applications.

Macromolecular Crowding by Polyethylene Glycol Reduces Protein Breathing.

Most efforts to understand macromolecular crowding focus on global (i.e., complete) unfolding, but smaller excursions, often called breathing, promote aggregation, which is associated with several diseases and the bane of pharmaceutical and commercial protein production. We used NMR to assess the effects of ethylene glycol (EG) and polyethylene glycols (PEGs) on the structure and stability of the B1 domain of protein G (GB1). Our data show that EG and PEGs stabilize GB1 differently. EG interacts with GB1 more strongly than PEGs, but neither affects the structure of the folded state. EG and 12000 g/mol PEG stabilize GB1 more than PEGs of intermediate size, but EG and smaller PEGs stabilize GB1 enthalpically while the largest PEG acts entropically. Our key finding is that PEGs turn local unfolding into global unfolding, and meta-analysis of published data supports this conclusion. These efforts provide knowledge that can be applied to improve biological drugs and commercial enzymes.

Efficient expression of fusion human epidermal growth factor in tobacco chloroplasts

BackgroundChloroplast transformation is a robust technology for the expression of recombinant proteins. Various types of pharmaceutical proteins including growth factors have been reported in chloroplasts via chloroplast transformation approach at high expression levels. However, high expression of epidermal growth factor (EGF) in chloroplasts with the technology is still unavailable.ResultsThe present work explored the high-level expression of recombinant EGF, a protein widely applied in many clinical therapies, in tobacco chloroplasts. In this work, homoplastic transgenic plants expressing fusion protein GFP-EGF, which was composed of GFP and EGF via a linker, were generated. The expression of GFP-EGF was confirmed by the combination of green fluorescent observation and Western blotting. The achieved accumulation of the recombinant fusion GFP-EGF was 10.21 ± 0.27% of total soluble proteins (1.57 ± 0.05 g kg− 1 of fresh leaf). The chloroplast-derived GFP-EGF was capable of increasing the cell viability of the NSLC cell line A549 and enhancing the phosphorylation level of the EGF receptor in the A549 cells.ConclusionThe expression of recombinant EGF in tobacco chloroplasts via chloroplast transformation method was achieved at considerable accumulation level. The attempt gives a good example for the application of chloroplast transformation technology in recombinant pharmaceutical protein production.

Double-Gene Copromoting Expression Analysis in tPA/GH Transgenic Goat Mammary Epithelial Cells and Thrombolytic Activity of tPA In Vitro.

Human tissue-plasminogen activator (tPA) is a thrombolytic drug widely used in the treatment of stroke, pulmonary thrombosis, acute myocardial infarction, and other thrombotic diseases. The double genes cointegrated into the organisms and cells can produce a synergistic effect, which will improve the expression level of the target gene. However, the study of the integration of the GH and tPA genes to improve the expression level of tPA has not yet been reported. In order to elucidate this, we generated monoclonal goat mammary epithelial cell lines with tPA/GH double-gene integration and analyzed the tPA expression level in single- and double-gene integrated cells. We selected the mammary gland-specific expressing vectors BLC14/tPA and BLC14/GH with the β-lactoglobulin gene as a regulatory sequence in our previous research. The tPA and GH genes were electronically cotransfected into goat mammary epithelial cells. Resistant cell lines were screened by G418, and transgenic monoclonal cell lines were confirmed by PCR. The tPA expression was induced by prolactin and detected in the cell induction solution after 48 h by ELISA and Western blotting. We detected the tPA biological activity in vitro by fibrin agarose plate assay (FAPA). The results showed that a total of 207 resistant monoclonal cells were obtained, including 126 cell lines with tPA monogenic integration and 51 cell lines with tPA/GH double-gene integration. The rate of double-gene integration was 24.6% (51/207). A total of 48 cells expressed tPA, of which 25.3% (19/75) cells expressed single gene, and 56.9% (29/51) cells expressed double genes. The concentration of tPA in single-gene-expressing cells was 8.0-64.0 μg/mL, and the tPA level in double-gene-expressing cells was significantly higher (200-7200 μg/mL). In addition, the tPA had a relatively strong in vitro thrombolytic activity determined by FAPA. The results showed that goat mammary epithelial cell lines with tPA/GH gene integration were successfully established by electrotransfection, and the expression level of tPA in double-gene integrated cell lines was significantly increased. This study provided a new way for the preparation of a transgenic goat and other animal with high tPA expression by somatic cell nuclear transfer. The findings also laid a foundation for efficient production of pharmaceutical proteins in transgenic animal mammary gland bioreactors in the future.

Potential of the Signal Peptide Derived from the PAS_chr3_0030 Gene Product for Secretory Expression of Valuable Enzymes in Pichia pastoris.

Pichia pastoris is widely used for the production of valuable recombinant proteins. An advantage of P. pastoris over other expression systems is that it secretes low levels of endogenous proteins, which facilitates the purification processes if the desired recombinant proteins are efficiently secreted into the culture medium. However, not all recombinant proteins can be successfully secreted by P. pastoris, especially enzymes that are located in intracellular compartments in their native hosts. Few studies have reported strategies for releasing recombinant proteins which cannot be secreted by standard protocols. Here, we investigated whether this challenge can be addressed using novel secretion leaders. Analysis of the secretome and transcriptome of P. pastoris indicated that the four genes with the highest protein-to-transcript ratios were EPX1, PAS_chr3_0030, SCW10, and UTH1, suggesting that their gene products contain efficient secretion leaders. Our data revealed that the signal peptide derived from the PAS_chr3_0030 gene product conferred secretion competence to certain industrial enzymes, e.g., a nitrilase of Alcaligenes faecalis ZJUTB10, a ribosylnicotinamide kinase of P. pastoris, and a glucose dehydrogenase of Exiguobacterium sibiricum. Therefore, the signal peptide derived from the PAS_chr3_0030 gene product represents a novel secretion sequence for the secretory expression of recombinant enzymes in P. pastoris. IMPORTANCE Although P. pastoris is widely used for the secretory production of pharmaceutical proteins, its successful applications in the secretory production of industrial enzymes are limited. The α-mating factor pre-pro leader is the most widely used secretion signal in P. pastoris, but numerous industrial enzymes cannot be secreted using it. The importance of this study is that we identified a signal peptide derived from the PAS_chr3_0030 gene product which conferred secretion competence to three-quarters of the enzymes tested. This signal peptide derived from the PAS_chr3_0030 gene product may facilitate the application of P. pastoris in industrial biocatalysis.

Compendium on Food Crop Plants as a Platform for Pharmaceutical Protein Production.

Tremendous advances in crop biotechnology related to the availability of molecular tools and methods developed for transformation and regeneration of specific plant species have been observed. As a consequence, the interest in plant molecular farming aimed at producing the desired therapeutic proteins has significantly increased. Since the middle of the 1980s, recombinant pharmaceuticals have transformed the treatment of many serious diseases and nowadays are used in all branches of medicine. The available systems of the synthesis include wild-type or modified mammalian cells, plants or plant cell cultures, insects, yeast, fungi, or bacteria. Undeniable benefits such as well-characterised breeding conditions, safety, and relatively low costs of production make plants an attractive yet competitive platform for biopharmaceutical production. Some of the vegetable plants that have edible tubers, fruits, leaves, or seeds may be desirable as inexpensive bioreactors because these organs can provide edible vaccines and thus omit the purification step of the final product. Some crucial facts in the development of plant-made pharmaceuticals are presented here in brief. Although crop systems do not require more strictly dedicated optimization of methodologies at any stages of the of biopharmaceutical production process, here we recall the complete framework of such a project, along with theoretical background. Thus, a brief review of the advantages and disadvantages of different systems, the principles for the selection of cis elements for the expression cassettes, and available methods of plant transformation, through to the protein recovery and purification stage, are all presented here. We also outline the achievements in the production of biopharmaceuticals in economically important crop plants and provide examples of their clinical trials and commercialization.

Development of a heterologous gene expression vector in plants based on an infectious clone of a tobravirus, pepper ringspot virus

AbstractHeterologous gene expression systems are important tools for biotechnology, especially due to the increasing demands for mass production of pharmaceutical proteins. In this study, we assessed the use of the infectious clone of a tobravirus, pepper ringspot virus (PepRSV), as a heterologous gene expression tool in Nicotiana benthamiana plants. The insert region of gene of interest was evaluated in the infectious clone of PepRSV using the reporter gene encoding the green fluorescent protein (GFP). The insertion of the GFP gene in the coat protein gene region resulted in a higher protein yield than the previous system. Then, the reporter gene was replaced by the xylanase gene, and enzymatically active xylanase was produced. This study provides an efficient tool for the production of heterologous proteins using a plant system.

Exploitation of the ribosomal protein L10 R98S mutation to enhance recombinant protein production in mammalian cells.

Mammalian cells are commonly used to produce recombinant protein therapeutics, but suffer from a high cost per mg of protein produced. There is therefore great interest in improving protein yields to reduce production cost. We present an entirely novel approach to reach this goal through direct engineering of the cellular translation machinery by introducing the R98S point mutation in the catalytically essential ribosomal protein L10 (RPL10‐R98S). Our data support that RPL10‐R98S enhances translation levels and fidelity and reduces proteasomal activity in lymphoid Ba/F3 and Jurkat cell models. In HEK293T cells cultured in chemically defined medium, knock‐in of RPL10‐R98S was associated with a 1.7‐ to 2.5‐fold increased production of four transiently expressed recombinant proteins and 1.7‐fold for one out of two stably expressed proteins. In CHO‐S cells, eGFP reached a 2‐fold increased expression under stable but not transient conditions, but there was no production benefit for monoclonal antibodies. The RPL10‐R98S associated production gain thus depends on culture conditions, cell type, and the nature of the expressed protein. Our study demonstrates the potential for using a ribosomal protein mutation for pharmaceutical protein production gains, and further research on how various factors influence RPL10‐R98S phenotypes can maximize its exploitability for the mammalian protein production industry.

Applications of CRISPR/Cas9 in a rice protein expression system via an intron-targeted insertion approach

The sugar starvation-inducible rice αAmy3 promoter and signal peptide are widely used to produce valuable recombinant proteins in rice suspension culture cells. Conventionally, the recombinant gene expression cassette is inserted into the genome at random locations by Agrobacterium- or particle bombardment-mediated transformation. CRISPR/Cas9 gene editing enables gene insertion at a precise target site in the genome. In this study the CRISPR/Cas9 approach was modified for intron-targeted insertion by adding an artificial 3′ splicing site upstream of the recombinant gene. Knock-in transgenic rice cell lines containing the recombinant GFP gene inserted in intron 1 of αAmy3 were generated. The endogenous αAmy3 promoter regulated recombinant gene expression and the αAmy3 signal peptide directed secretion of the recombinant GFP protein into the culture medium. In addition, the recombinant GFP protein was localized in amyloplasts, identical to the subcellular localization of endogenous αAmy3 reported previously. This modified CRISPR/Cas9 knock-in approach is simple and highly efficient, and the recombinant gene insertion frequency attained 12.5%. The approach can be applied in the production of pharmaceutical proteins in rice suspension cell cultures. The high efficiency of the GFP reporter gene knock-in method and the maintenance of target gene behavior also make the strategy applicable to endogenous gene functional studies in rice.

Leishmania Parasite: the Impact of New Serum-Free Medium as an Alternative for Fetal Bovine Serum

Background:Flagellated protozoan of the genus Leishmania is the causative agent of vector-borne parasitic diseases of leishmaniasis. Since the production of recombinant pharmaceutical proteins requires the cultivation of host cells in a serum-free medium, the elimination of FBS can improve the possibility of large-scale culture of Leishmania parasite. In the current study, we aimed at evaluating a new serum-free medium in Leishmania parasite culture for future live Leishmania vaccine purposes. Methods:Recombinant L. tarentolae secreting PpSP15-EGFP and wild type L. major were cultured in serum-free (CSFM) and serum-supplemented medium. The growth rate, protein expression, and infectivity of cultured parasites in both conditions was then evaluated and compared. Results:Diff-Quick staining and epi-fluorescence microscopy examination displayed the typical morphology of L. major and L. tarentolae-PpSP15-EGFP promastigote grown in CSFM medium. The amount of EGFP expression was similar in CSMF medium compared to M199 supplemented with 5% FBS in flow cytometry analysis of L. tarentolae-PpSP15-EGFP parasite. Also, a similar profile of PpSP15-EGFP proteins was recognized in Western blot analysis of L. tarentolae-PpSP15-EGFP cultured in CSMF and the serum-supplemented medium. Footpad swelling and parasite load measurements showed the ability of CSFM medium to support the L. major infectivity in BALB/C mice. Conclusion:This study demonstrated that CSFM can be a promising substitute for FBS supplemented medium in parasite culture for live vaccination purposes.

Transient Production of Human β-Glucocerebrosidase With Mannosidic-Type N-Glycan Structure in Glycoengineered Nicotiana benthamiana Plants.

Gaucher disease is an inherited lysosomal storage disorder caused by a deficiency of functional enzyme β-glucocerebrosidase (GCase). Recombinant GCase has been used in enzyme replacement therapy to treat Gaucher disease. Importantly, the terminal mannose N-glycan structure is essential for the uptake of recombinant GCase into macrophages via the mannose receptor. In this research, recombinant GCase was produced using Agrobacterium-mediated transient expression in both wild-type (WT) and N-acetylglucosaminyltransferase I (GnTI) downregulated Nicotiana benthamiana (ΔgntI) plants, the latter of which accumulates mannosidic-type N-glycan structures. The successfully produced functional GCase exhibited GCase enzyme activity. The enzyme activity was the same as that of the conventional mammalian-derived GCase. Notably, N-glycan analysis revealed that a mannosidic-type N-glycan structure lacking plant-specific N-glycans (β1,2-xylose and α1,3-fucose residues) was predominant in all glycosylation sites of purified GCase produced from ΔgntI plants. Our research provides a promising alternative plant line as a host for the production of recombinant GCase with a mannosidic-type N-glycan structure. This glycoengineered plant might be applicable to the production of other pharmaceutical proteins, especially mannose receptor targeted protein, for therapeutic uses.

Wolffia arrhiza as a promising producer of recombinant hirudin.

The production of recombinant proteins in transgenic plants is becoming an increasingly serious alternative to classical biopharming methods as knowledge about this process grows. Wolffia arrhiza, an aquatic plant unique in its anatomy, is a promising expression system that can grow in submerged culture in bioreactors. In our study 8550 explants were subjected to Agrobacterium-mediated transformation, and 41 independent hygromycin-resistant Wolffia lines were obtained, with the transformation efficiency of 0.48%. 40 of them contained the hirudin-1 gene (codon-optimized for expression in plants) and were independent lines of nuclear-transformed Wolffia, the transgenic insertion has been confirmed by PCR and Southern blot analysis. We have analyzed the accumulation of the target protein and its expression has been proven in three transgenic lines. The maximum accumulation of recombinant hirudin was 0.02% of the total soluble protein, which corresponds to 775.5 ± 111.9ngg-1 of fresh weight of the plant. The results will be used in research on the development of an expression system based on Wolffia plants for the production of hirudin and other recombinant pharmaceutical proteins.

Genome Editing of Saccharomyces Cerevisiae Using CRISPR-Cas9 System

Saccharomyces cerevisiae is an important yeast has been exploited for a long time to produce alcohol or bread. Moreover, genetically engineered S. cerevisiae cells continue to be used as cell factories for production of biofuels, pharmaceutical proteins and food additives. Genetically modified strain of S. cerevisiae created using traditional methods is laborious and time consuming. Recently, originally an immune system in archaea and bacteria, Clustered regularly interspaced short palindromic repeats “CRISPR” and CRISPR-associated “Cas” have been used exploited as a flexible tool for genome editing. Until now, this tool has been applied to many organisms including yeast. Here, we review the importance of S. cerevisiae as an industrial platform and the use of CRISPR/Cas system and its applications in research and industry of this yeast.

Pharmaceutical protein solids: Drying technology, solid-state characterization and stability.

Despite the boom in biologics over the past decade, the intrinsic instability of these large molecules poses significant challenges to formulation development. Almost half of all pharmaceutical protein products are formulated in the solid form to preserve protein native structure and extend product shelf-life. In this review, both traditional and emerging drying techniques for producing protein solids will be discussed. During the drying process, various stresses can impact the stability of protein solids. However, understanding the impact of stress on protein product quality can be challenging due to the lack of reliable characterization techniques for biological solids. Both conventional and advanced characterization techniques are discussed including differential scanning calorimetry (DSC), solid-state Fourier transform infrared spectrometry (ssFTIR), solid-state fluorescence spectrometry, solid-state hydrogen deuterium exchange (ssHDX), solid-state nuclear magnetic resonance (ssNMR) and solid-state photolytic labeling (ssPL). Advanced characterization tools may offer mechanistic investigations into local structural changes and interactions at higher resolutions. The continuous exploration of new drying techniques, as well as a better understanding of the effects caused by different drying techniques in solid state, would advance the formulation development of biological products with superior quality.

CALLUS INDUCTION AND EXTRACTING MEDICAL MATERIALS

In recent year, the importance of plants has been Known as a source of pharmaceuticals, due to many factors, the most important of which is the rapid progress in bio molecular technologies where methods have been developed for the transfer of genes and the use of the plant as an alternative source in the production of pharmaceutical proteins, including vaccines, antibodies, enzymes, bioactive compounds and free of viral and pathogens. Therefore, the technology of tissue culture and the generation of cells and plant organs outside the living body is an indispensable tool in the production of medicinal materials derived from plants, and this method has significant productivity benefits in terms of quantity, quality and production controlled without restricting natural factors such as geographical location, seasonal changes, or environmental stresses.

Retrotransposon-mediated Gene Transfer for Animal Cells

Gene delivery methods for animal cells are one of the most important tools in biotechnology fields such as pharmaceutical protein production, generation of transgenic animals and gene therapy. Because retrotransposons can move their own sequences to new genomic locations by a “copy-and-paste” process known as retrotransposition, we attempted to develop a novel gene transfer system based on retrotransposon. A full-length long interspersed element-1 (LINE-1) contains a 5’ untranslated region (5’UTR), two non-overlapping open reading frames (ORFs) separated by a short inter-ORF sequence, and a 3’UTR terminating in an adenosine-rich tract. We constructed a LINE-1 vector plasmid including components necessary for retrotransposition. An intron-disruptedNeoreporter gene and a scFv-Fc expression unit under the control of CMV promoter were added into 3’UTR in order to evaluate retrotransposition and express scFv-Fc. CHO-K1 cells transfected with the plasmids were screened with G418. The established cell clones produced scFv-Fc proteins in the culture medium. To control retrotransposition steadily, we also established retrotransposon systems that supply ORF2 or ORF1–2 separately. Genomic PCR analysis revealed that transgene sequences derived from the LINE-1 vector were positive in all clones. All the clones tested produced scFv-Fc in the culture medium.

Engineering Bacillus subtilis Cells as Factories: Enzyme Secretion and Value-added Chemical Production

Bacillus subtilis has been studied for more than half a century, ever since the dawn of molecular biology, as a representative Gram-positive bacterium and cell factory. Two characteristic capacities of B. subtilis, namely its natural competence for DNA uptake and high-level enzyme secretion, have been investigated and exploited intensively during these long years. As a consequence, this bacterium has evolved into an excellent platform for synthetic biological research and development. In this review, we outline basic concepts for B. subtilis cell factory engineering, and we describe several examples of its applications in the production of proteins and high-value metabolites. In particular, we highlight engineering approaches that can make the already very efficient Bacillus protein secretion pathways even more efficient for the production of enzymes and pharmaceutical proteins. We further showcase examples of metabolic engineering in B. subtilis based on synthetic biology principles to produce various high-value or health-promoting substances, especially inositol stereoisomers. We conclude that the versatile traits of B. subtilis, combined with multi-omics approaches and rapidly developing technologies for genome engineering and high-throughput screening enable us to modify and optimize this bacterium’s metabolic circuits to deliver compounds that are needed for a green and sustainable society as well as a healthy population.

Fusion of a Novel Native Signal Peptide Enhanced the Secretion and Solubility of Bioactive Human Interferon Gamma Glycoproteins in Nicotiana benthamiana Using the Bamboo Mosaic Virus-Based Expression System.

Plant viruses may serve as expression vectors for the efficient production of pharmaceutical proteins in plants. However, the downstream processing and post-translational modifications of the target proteins remain the major challenges. We have previously developed an expression system derived from Bamboo mosaic virus (BaMV), designated pKB19, and demonstrated its applicability for the production of human mature interferon gamma (mIFNγ) in Nicotiana benthamiana. In this study, we aimed to enhance the yields of soluble and secreted mIFNγ through the incorporation of various plant-derived signal peptides. Furthermore, we analyzed the glycosylation patterns and the biological activity of the mIFNγ expressed by the improved pKB19 expression system in N. benthamiana. The results revealed that the fusion of a native N. benthamiana extensin secretory signal (SSExt) to the N-terminal of mIFNγ (designated SSExt mIFNγ) led to the highest accumulation level of protein in intracellular (IC) or apoplast washing fluid (AWF) fractions of N. benthamiana leaf tissues. The addition of 10 units of ‘Ser-Pro’ motifs of hydroxyproline-O-glycosylated peptides (HypGPs) at the C-terminal end of SSExt mIFNγ (designated SSExt mIFNγ(SP)10) increased the solubility to nearly 2.7- and 1.5-fold higher than those of mIFNγ and SSExt mIFNγ, respectively. The purified soluble SSExt mIFNγ(SP)10 protein was glycosylated with abundant complex-type N-glycan attached to residues N56 and N128, and exhibited biological activity against Sindbis virus and Influenza virus replication in human cell culture systems. In addition, suspension cell cultures were established from transgenic N. benthamiana, which produced secreted SSExt mIFNγ(SP)10 protein feasible for downstream processing. These results demonstrate the applicability of the BaMV-based vector systems as a useful alternative for the production of therapeutic proteins, through the incorporation of appropriate fusion tags.

Identification, repair and characterization of a benzyl alcohol-inducible promoter for recombinant proteins overexpression in Corynebacterium glutamicum

Corynebacterium glutamicum is an important industrial organism for the production of a variety of biological commodities. We discovered a promoter encoded by the gene NCgl2319 in C. glutamicum, which could be induced by benzyl alcohol, could be used as an efficient tunable expression system. In initial attempts, this promoter failed to function in a recombinant expression system. This was remedied by extending the original genetic context of the promoter, generating a new version Pcat-B. The Pcat-B transcription initiation site, its critical active regions, and its effect of inducers were fully characterized resulting in tunable expression. This approach proved to be very efficient in producing a pharmaceutical protein, N-terminal pro-brain natriuretic peptide (NT-proBNP). Production of approximately 440.43 mg/L NT-proBNP was achieved with the Pcat-B expression system demonstrating its application for controllable pharmaceutical protein production in C. glutamicum.